Growth hormone (GH) is an important endocrine hormone that has effects on growth, maturation, and metabolism. GH interacts with specific receptors (GHRs) on various target tissues. It is believed that signals transduced via the GHR constitute the initiation of GH action. Recent evidence indicates that an early event in GHR signalling is the activation of a protein tyrosine kinase activity with resultant tyrosine phosphorylation of the receptor itself as well as a number of other cellular proteins. Since the GHR cDNA does not encode a recognizable kinase domain, it presumed that the kinase(s) activated by GH treatment are not intrinsic to the receptor, but rather are separate proteins that physically or functionally interact with GHR in response to the hormone. Preliminary data from studies of the human B-lymphoblastoid line, IM-9, reveal the acute GH-induced augmentation of in vitro tyrosine autokinase activity of two members of the src- family of non-receptor tyrosine kinases, lyn and fyn. Further, other preliminary results indicate that the GHRs that undergo GH-induced tyrosine phosphorylation in these cells are disulfide linked to either themselves or to another similarly sized protein. In an attempt to further understand the molecular physiology of GH action with regard to tyrosine kinase activation, experiments will build on these preliminary results in accomplishing the following four objectives: 1) An attempt will be made to understand which protein tyrosine kinase(s) are activated upon GH treatment. Further use of in vitro kinase assays as well as immunoprecipitation and immunoblotting experiments with anti-src-family specific antisera will be pursued. 2) Studies will be directed at determining whether several known proteins relevant to signal transduction, including extracellular signal-regulated kinases (ERKs), the raf protooncogene product, and src-family kinases are tyrosine phosphorylated in response to GH. 3) The potential physical association of GH-activated tyrosine kinase(s) with the GHR will be studied. Anti-GHR antibodies as well as bacterially-expressed fusion proteins incorporating the GHR cytoplasmic domain will be used as affinity probes in immunoblotting and in vitro kinase assays of GH-stimulated cell lysates. 4) Studies regarding the in vivo topology of the GHR will be performed as an extension of preliminary findings that a pool of GHRs that is tyrosine phosphorylated in response to GH exists in a disulfide-linked form. These studies will involve determining the structure of labelled GHRs in the basal and GH-stimulated state by immunoprecipitation and two-dimensional electrophoresis techniques. These studies should provide insight into the molecular relationship between activated GHRs and important cellular kinases and may shed light on the specific targets for these kinases with regard to GH action. Results obtained may also relate to the mechanisms of action of receptors for other cytokines and hematopoietins important in health and disease.
